Synthetic Systems for Teaching and Learning

Winston Retreat

June 25th, 2007

Natalie Kuldell

Regulating RNA degradation in yeast

mitochondria

Undergraduate teaching with SAGA

deletions

Unreal Irrational

Why hack the yeast mitochondria?

“ (we often) imagine the mitochondrion as a lonely participant in the cell, working tirelessly to produce the energy required for life.”

McBride et. al. Curr Biol 2006

http://grocs.dmc.dc.umich.edu/gallery/organelle/Interface2

Other mt functions

• coordinates with nuclear gene expression (disease/aging)

• spatially isolated enzymatic reaction center

• viability on nonfermentable carbon sources

? =

Hacking yeast mitochondria

Wish list (incomplete)

1. Orthogonal draw from different pools of reagents

2. Decoupled run system independent of growth rate

3. Generic run same system in different chassis

4. Tunable vary operation at will

Current contents: in mt from mt

mt genome includes

• 8 protein coding genes

7 oxphos, 1 riboprot

• 2 rRNAs

• 24 tRNAs

mt promoters TATAAGTA (+1)

mt RNAP RPO41 = catalytic subunit MTF1 = specificity factor

nuclear-encoded

Targeted mtRNA degradation

Part 3: dsRNase

Part 1: mRNA target

e.g. mtGFP

Part 2: guide RNA

Catala et al, MCB (2004) 15:3015

Snapshot of wild type role for Rnt1

• Localized to the nucleus even when overexpressed

Snapshot of wild type role for Rnt1

• Processes some noncoding RNAs (U2 snRNA, U3 snoRNPs)

• Localized to the nucleus even when overexpressed

Henras et al.RNA (2004) 10: 1572

Snapshot of wild type role for Rnt1

• Processes some noncoding RNAs (U2 snRNA, U3 snoRNPs)

• Localized to the nucleus even when overexpressed

• Processes some coding RNA, e.g. Mig2

Ge et al, Current Biology (2005) 15:140

Catala et al, MCB (2004) 15:3015

Snapshot of wild type role for Rnt1

• Processes some noncoding RNAs (U2 snRNA, U3 snoRNPs)

• Localized to the nucleus even when overexpressed

• Processes some coding RNA, e.g. Mig2

• Needed for normal cell cycle progression

tTA

CMV

2x tetO

CYC1

pRS41n

modified RNT1

Expression vector for mitochondrial Rnt1

Expression vector for mitochondrial Rnt1

pRS41n

RNT1

signal sequence + epitope tag

∆NLS (11 aa)

∆NLS in Henras et al RNA (2004) 10:1572

Initial experiments with mtRnt1

1. Expression? by Western with epitope Ab

2. Phenotypes? Respiration, growth, existing markers

3. Overall? Microarray wt vs mtRnt

Targeted mtRNA degradation

Part 3: dsRNase

Part 1: mRNA target

e.g. mtGFP

Part 2: guide RNA

Protein import into mitochondria

Pfanner and Geissler Nat Rev

(2001) 2:339

RNA import into mitochondria“poorly understood”/”mechanisms appear to differ”

RNA import into mitochondria“poorly understood”/”mechanisms appear to differ”

~all mt tRNAs encoded on

nuclear genome

RNA receptor (“RIC”) in mt membrane Entelis et al Gene Engineering:

Principles and Methods (2001) 24:191

RNA import into mitochondria“poorly understood”/”mechanisms appear to differ”

no mt tRNAs encoded by mt

RIC + ytRNA--> repair mt defect in

human cell line

Mahata et al Science (2006) 314:471

RNA import into mitochondria“poorly understood”/”mechanisms appear to differ”

all but one tRNA encoded on mt

genome

import depends on protein import

Specialized import into mitochondria

protein:RNA conjugate

Piggyback on tRNA import

Bind to mtRNA binding protein

Regulating RNA degradation in yeast

mitochondria

Undergraduate teaching with SAGA

deletions

Unreal Irrational

Expression Engineering Experiment

Day 1 Day 2 Day 3

Day 4Day 5Day 6

RT

Subunit Deleted? ADA3 1/1

GCN5 1/1

SPT3 (3 groups) 3/3

SPT8 (3 groups) 0/3

UBP8 (2 groups) 2/2

SUS1 (2 groups) 2/2

FY2068 A ura3-52 his3∆200 leu2∆1 lys2-128

Subunit Deleted? ADA3 1/1

GCN5 1/1

SPT3 (3 groups) 3/3

SPT8 (3 groups) 0/3

UBP8 (2 groups) 2/2

SUS1 (2 groups) 2/2

FY2068 A ura3-52 his3∆200 leu2∆1 lys2-128

NY389

ura3-52 his4-917leu3∆1 trp1-63 spt8∆320::LEU2

Day 3

wt/sus1∆

Andrew Ji and Kate Broadbent, W/F Team Blue, 20.109 Spring ‘07

wt/sgf73∆

teacher

Follow-up with microarray

Follow-up with spot tests

Hi Natalie,I've attached my rewrite. Thanks! See you tomorrow,AndrewP.S. This was one of the most time-consuming assignments I've ever had to do, yet it was easily the most fun and rewarding thing I've ever accomplished for any school-related project.

Follow-up with spot tests

Andrew Ji and Kate Broadbent

May 10, 2007

Sus1’s role in SAGA-dependent gene motility, transcription, and

expression under different cellular conditions

From: Neal Lerner <nlerner@MIT.EDU>Subject: Re: 109 writing assignmentDate: Thu, 11 Jan 2007 11:07:34 -0500To: natalie kuldell <nkuldell@mit.edu>

Natalie, as I prepare to give a writing-across-the-curriculum talk next week, I came across this quote from John Bean: WAC is about creating opportunities for students to have an "authentic desire to converse with interested readers about real ideas." Now, in most school settings that's pretty darn hard to achieve, but I think when students have the chance to write/talk about lab work and ideas they find interesting (as in 20.109), we have a shot at it.

See you on the 22nd.

Neal

the end

Current contents: in mt from nucleus

nuclear genome sends

• ~750 proteins to mt

87 of these are putative proteins of no known function